Methods Predicting Risk of an Adverse Clinical Outcome

ABSTRACT

Provided are methods for evaluating the risk of an adverse clinical outcome in a subject, deciding whether to discharge or continue treating a subject (e.g., treatment on an inpatient basis), or to initiate or terminate treatment, selecting a subject for participation in a clinical study, and selecting a therapeutic treatment for a subject that include determining a level of ST2 in a biological sample from the subject and determining a level of galectin-3 in a biological sample from the subject. Kits are also provided that contain an antibody that specifically binds to ST2, an antibody that specifically binds to galectin-3, and instructions for using the kit to evaluate the risk of an adverse clinical outcome in a subject, to decide whether to discharge or continue treating a subject (e.g., treatment on an inpatient basis) or to initiate or terminate treatment, to select a subject for participation in a clinical study, and/or to select treatment for a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/422,574, filed Mar. 16, 2012, which claimspriority to U.S. Provisional Patent Application Ser. No. 61/453,782,filed Mar. 17, 2011, the contents of each of which are incorporatedherein by reference in their entirety.

TECHNICAL FIELD

Described herein are methods for the determining the risk of an adverseclinical outcome in a subject, selecting a therapeutic treatment for asubject, and selecting patients for participation in a clinical study.

SUMMARY

The present invention is based, at least in part, on the surprisingdiscovery that the presence of an elevated level of galectin-3 or thepresence of an elevated level of ST2 (also known as Interleukin 1Receptor Like-1 (IL1RL1)) indicates a subject with an increased risk ofan adverse clinical outcome (ACO), and the presence of both an elevatedlevel of galectin-3 and an elevated level of ST2 indicates a subjectwith a greatly increased risk of an ACO. Thus, in some aspects, themethods described herein include determining the levels of galectin-3and ST-2 in a subject, and, optionally, determining the levels of one ormore (e.g., two, three, or four) of proANP, NT-pro-ANP, ANP, proBNP,NT-proBNP, BNP, troponin, CRP, creatinine, Blood Urea Nitrogen (BUN),liver function enzymes, albumin, and bacterial endotoxin in the subject.These methods can be used to determine the risk of an ACO, decidewhether to discharge or to initiate, continue, or terminate treatment ofa subject (e.g., treatment on an inpatient basis), select a subject forparticipation in a clinical study, or select a therapeutic treatment fora subject.

Accordingly, provided herein are methods for evaluating the risk of anACO in a subject that include the steps of: (a) determining a level ofST2 in a biological sample (e.g., serum) from the subject, and (b)determining a level of galectin-3 in a biological sample (e.g., serum)from the subject, where the subject's levels of ST2 and galectin-3relative to a reference levels of ST2 and galectin-3 indicate thesubject's risk of an ACO. In some embodiments of these methods, thepresence of an elevated level of ST2 or the presence of an elevatedlevel of galectin-3 indicates an increased risk of an ACO, and thepresence of both an elevated level of ST2 and an elevated level ofgalectin-3 indicates a greatly increased risk of an ACO. In someembodiments of these methods, the presence of both a non-elevated levelof ST2 and a non-elevated level of galectin-3 indicates a reduced riskof an ACO. In some embodiments of these methods, the risk of an ACO iswithin 1 year or within 30 days.

Also provided are methods for deciding whether to discharge or initiate,terminate, or continue treating a subject (e.g., treating on aninpatient basis that include the steps of: (a) determining a level ofST2 in a biological sample (e.g., serum) from the subject, and (b)determining a level of galectin-3 in a biological sample (e.g., serum)from the subject, where the subject's levels of ST2 and galectin-3relative to reference levels of ST2 and galectin-3 determine whether thesubject should be discharged, receive continued treatment (e.g.,treatment on an inpatient basis), or whether treatment should beinitiated or terminated. In some embodiments of these methods, thepresence of an elevated level of ST2 or the presence of an elevatedlevel of galectin-3 indicates that the subject should receive continuedtreatment (e.g., treatment on an inpatient basis) or that treatmentshould be initiated, and the presence of both an elevated ST2 level andan elevated level of galectin-3 strongly indicates that the subjectshould receive continued treatment (e.g., treatment on an inpatientbasis) or that treatment should be initiated. In some embodiments ofthese methods, the presence of both a non-elevated level of ST2 and anon-elevated level of galectin-3 indicates that the subject should bedischarged, receive treatment on an outpatient basis, or that treatmentshould be terminated.

Also provided are methods of selecting a subject for participation in aclinical study that include the steps of: (a) determining a level of ST2in a biological sample (e.g., serum) from the subject, and (b)determining a level of galectin-3 in a biological sample (e.g., serum)from the subject, and selecting the subject for participation in aclinical study if the subject's levels of ST2 and galectin-3 relative toreference levels of ST2 and galectin-3 indicate that the subject shouldbe selected for participation in a clinical study. In some embodimentsof these methods, the presence of an elevated level of ST2 or thepresence of an elevated level of galectin-3 indicates that the subjectshould be selected for participation in a clinical study, and thepresence of both an elevated level of ST2 and an elevated level ofgalectin-3 strongly indicates that the subject should be selected forparticipation in a clinical study. In some embodiments of these methods,the presence of a non-elevated level of ST2 and/or the presence of anon-elevated level of galectin-3 indicates that the subject should beexcluded from participation in a clinical study.

Also provided are methods for selecting a therapeutic treatment for asubject that include the steps of: (a) determining a level of ST2 in abiological sample (e.g., serum) from the subject, and (b) determining alevel of galectin-3 in a biological sample (e.g., serum) from thesubject, where the subject's levels of ST2 and galectin-3 relative toreference levels of ST2 and galectin-3 are used to select a therapeutictreatment for the subject. In some embodiments of these methods, thepresence of an elevated level of ST2 or the presence of an elevatedlevel of galectin-3 is used to select the therapeutic treatment for thesubject, and the presence of both an elevated level of ST2 and anelevated level of galectin-3 is predominantly used to select thetherapeutic treatment for the subject. In some embodiments of thesemethods, the presence of a non-elevated level of ST2 and/or the presenceof a non-elevated level of galectin-3 is used to select the therapeutictreatment for the subject. The therapeutic treatment may be selectedfrom the group of: nitrates, calcium channel blockers, diuretics,thrombolytic agents, digitalis, renin-angiotensin-aldosterone system(RAAS) modulating agents (e.g., beta-adrenergic blocking agents (e.g.,alprenolol, bucindolol, carteolol, carvedilol, labetalol, nadolol,penbutolol, pindolol, propranolol, sotalol, timolol, cebutolol,atenolol, betaxolol, bisoprolol, celiprolol, esmolol, metoprolol, andnebivolol), angiotensin-converting enzyme inhibitors (e.g., benazepril,captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril,quinapril, ramipril, and trandolapril), aldosterone antagonists (e.g.,spironolactone, eplerenone, canrenone (canrenoate potassium), prorenone(prorenoate potassium), and mexrenone (mexrenoate potassium)), renininhibitors (e.g., aliskiren, remikiren, and enalkiren), and angiotensinII receptor blockers (e.g., valsartan, telmisartan, losartan,irbesartan, and olmesartan)), and cholesterol-lowering agents (e.g., astatin).

In some embodiments of all of the methods described herein, the ACO maybe rehospitalization, recurrence of one or more (e.g., two, three, orfour) physical symptoms associated with a disease state, an increase inthe severity of one or more (e.g., two, three, or four) physicalsymptoms associated with a disease state, an increase in the frequencyof one or more (e.g., two, three, or four) physical symptoms associatedwith a disease state, mortality (e.g., mortality due to CVD), admissionto a health care facility (e.g., a hospital or assisted care facility),or organ transplant (e.g., heart transplant). In some embodiments, thedisease state may be angina, cardiovascular disease, and heart failure.In the above methods, the rehospitalization or admission may be forcardiovascular disease.

In any of the above aspects, the subject may have been diagnosed with acardiac disease (e.g., heart failure, heart attack, coronary arterydisease, cardiovascular disease, acute coronary syndrome, and angina),inflammation, stroke, renal failure, obesity, high cholesterol, and/ordyslipidemia. In some embodiments of the above methods, the subject maybe undiagnosed, normal, or apparently healthy. In some examples of allof the above methods, the sample may be serum, blood, or plasma. In someexamples of the above methods, the sample in step (a) and the sample instep (b) are obtained from the subject at the same time.

In any of the above aspects, the subject may have an elevated BMI, a BMIof 25-29, a BMI of ≧30, or renal insufficiency. In further examples ofany of the above methods, the reference level of ST2 is a level of ST2in a subject that does not have high risk cardiovascular disease; thereference level of ST2 is a threshold level of ST2; the reference levelof galectin-3 is a level of galectin-3 in a subject that does not havehigh risk cardiovascular disease or does not have galectin-3 positivecardiovascular disease; or the reference level of galectin-3 is a levelof galectin-3 before or after onset of one or more (e.g., two, three,four, or five) disease (e.g., cardiac disease (e.g., heart failure,coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension)symptoms; before or after diagnosis with disease (e.g., cardiac disease(e.g., heart failure, coronary artery disease, cardiovascular disease,acute coronary syndrome, and angina), renal insufficiency, stroke, orhypertension); before or after therapeutic treatment for a disease(e.g., cardiac disease (e.g., heart failure, coronary artery disease,cardiovascular disease, acute coronary syndrome, and angina), renalinsufficiency, stroke, or hypertension); or at a different time pointduring therapeutic treatment (e.g., inpatient or outpatient treatment)for a disease (e.g., cardiac disease (e.g., heart failure, coronaryartery disease, cardiovascular disease, acute coronary syndrome, andangina), renal insufficiency, stroke, or hypertension); or before andafter a cardiac event (e.g., a myocardial infarction).

In some embodiments of all of the above methods, the method furtherincludes determining the level of one or more (e.g., two, three, orfour) additional markers in the subject (e.g., proANP, NT-proANP, ANP,proBNP, NT-proBNP, BNP, troponin, CRP, creatinine, Blood Urea Nitrogen(BUN), liver function enzymes, albumin, and bacterial endotoxin).

Also provided are kits containing an antibody that specifically binds toST2, an antibody that specifically binds to galectin-3, and instructionsfor using the kit in any of the methods described herein.

By the term “adverse clinical outcome” or “ACO” is meant an increase(e.g., by at least one, two, three, or four) in the number of symptomsor the severity or frequency of one of more (e.g., two, three, four, orfive) symptoms in a subject, death, or therapeutic treatment that isnecessitated by the increase (e.g., by at least one, two, three, orfour) in the number or the severity or frequency of one or more (e.g.,two, three, four, or five) symptoms in a subject. Non-limiting examplesof an ACO include rehospitalization, recurrence of one or more (e.g.,two, three, four, or five) physical symptoms associated with a diseasestate (e.g., cardiovascular disease), an increase in the severity of oneor more (e.g., two, three, four, or five) physical symptoms associatedwith a disease state, an increase in the frequency of one or more (e.g.,two, three, four, or five) physical symptoms associated with a diseasestate, mortality (e.g., mortality from a cardiovascular disease),admission to a health care facility (e.g., a hospital or assisted carefacility), or organ transplant (e.g., heart transplant). The symptomsmay be associated with a specific disease state, such a cardiac disease(e.g., heart failure, heart attack, coronary artery disease,cardiovascular disease, acute coronary syndrome, and angina),inflammation, stroke, renal failure, obesity, high cholesterol, ordyslipidemia.

By the term “ST2” or “soluble ST2” is meant a soluble protein containinga sequence at least 90% identical (e.g., at least 95%, 96%, 97%, 98%,99%, or 100% identical) to NCBI Accession No. NP_(—)003847.2 (SEQ IDNO: 1) or a nucleic acid containing a sequence at least 90% identical(e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to NCBIAccession No. NM_(—)003856.2 (SEQ ID NO: 2).

By the term “galectin-3” or “gal-3” is meant a protein containing asequence at least 90% identical (e.g., at least 95%, 96%, 97%, 98%, 99%,or 100% identical) to NCBI Accession No. NP_(—)001170859 (SEQ ID NO: 3),a protein containing a sequence at least 90% identical (e.g., at least95%, 96%, 97%, 98%, 99%, or 100% identical) to NCBI Accession No.NP_(—)002297 (SEQ ID NO: 4), a nucleic acid containing a sequence atleast 90% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%identical) to NCBI Accession No. NM_(—)001177388.1 (SEQ ID NO: 5), anucleic acid containing a sequence at least 90% identical (e.g., atleast 95%, 96%, 97%, 98%, 99%, or 100% identical) to NCBI Accession No.NM_(—)002306.3 (SEQ ID NO: 6).

By the term “elevated” or “elevation” is meant a statisticallysignificant difference (e.g., an increase of at least 5%, 10%, 20%, 25%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,120%, 140%, 160%, 180%, 200%, 220%, 240%, 260%, 280%, or 300% increase)in a determined or measured level (e.g., a galectin-3 or ST2 protein ornucleic acid level) compared to a reference level (e.g., a level ofgalectin-3 in a subject not having high risk CVD or not havinggalectin-3 positive cardiovascular disease, a threshold level ofgalectin-3, a level of ST2 in a subject not having CVD, and a thresholdlevel of ST2). The reference level of ST2 or galectin-3 may be a proteinor nucleic acid level. Additional reference levels of ST2 and galectin-3are described herein.

By the term “health care facility” is meant a location were a subjectmay receive medical care from a health care professional (e.g., a nurse,a physician, or a physician's assistant). Non-limiting examples ofhealth care facilities include hospitals, clinics, and assisted carefacilities (e.g., a nursing home).

By the term “reference level” is meant a threshold level or a level in acontrol subject or control patient population. A reference level willdepend on the assay performed and can be determined by one of ordinaryskill in the art. A reference level may be a baseline level or a levelin the same patient measured at an earlier or later point in time. Somenon-limiting examples of reference levels of ST2 include the level ofST2 in a subject that: does not have high risk CVD, does not have renalfailure, or has a BMI under 25. Additional control patient populationsare described herein. Additional examples of reference levels of ST2include threshold levels of ST2. Non-limiting examples of referencelevels of ST2 are known in the art and are described herein.

In some embodiments, the ratio of two ST2 levels in a subject iscompared to a reference level that is a ratio of ST2 levels measured ina subject (e.g., any of the control subjects described herein or thesame subject), for example, a reference level may be a ratio of thelevels of ST2 before and after onset of one or more (e.g., two, three,four, or five) disease (e.g., cardiac disease (e.g., heart failure,coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension)symptoms; a ratio of the levels of ST2 before and after diagnosis withdisease (e.g., cardiac disease (e.g., heart failure, coronary arterydisease, cardiovascular disease, acute coronary syndrome, and angina),renal insufficiency, stroke, or hypertension); a ratio of the levels ofST2 before and after therapeutic treatment for a disease (e.g., cardiacdisease (e.g., heart failure, coronary artery disease, cardiovasculardisease, acute coronary syndrome, and angina), renal insufficiency,stroke, or hypertension); a ratio of the ST2 levels at two differenttime points during therapeutic treatment (e.g., inpatient or outpatienttreatment) for a disease (e.g., cardiac disease (e.g., heart failure,coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension); ora ratio of the ST2 levels before and after a cardiac event (e.g., amyocardial infarction).

Non-limiting examples of reference levels of galectin-3 include thelevel of galectin-3 in a subject that: does not have high risk CVD, doesnot have galectin-3 positive cardiovascular disease, does not have renalfailure, or has a BMI under 25. Further control patient populations andthreshold levels for a galectin-3 control are described herein.Additional non-limiting examples of reference levels of galectin-3include threshold levels of galectin-3. Non-limiting examples ofreference levels of galectin-3 are known in the art and are describedherein.

Additional examples of a reference level of galectin-3 is a level ofgalectin-3 before or after onset of one or more (e.g., two, three, four,or five) disease (e.g., cardiac disease (e.g., heart failure, coronaryartery disease, cardiovascular disease, acute coronary syndrome, andangina), renal insufficiency, stroke, or hypertension) symptoms; a levelof galectin-3 before or after diagnosis with disease (e.g., cardiacdisease (e.g., heart failure, coronary artery disease, cardiovasculardisease, acute coronary syndrome, and angina), renal insufficiency,stroke, or hypertension); a level of galectin-3 before or aftertherapeutic treatment for a disease (e.g., cardiac disease (e.g., heartfailure, coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension); ora level of galectin-3 at a different time point during therapeutictreatment (e.g., inpatient or outpatient treatment) for a disease (e.g.,cardiac disease (e.g., heart failure, coronary artery disease,cardiovascular disease, acute coronary syndrome, and angina), renalinsufficiency, or hypertension); or before and after a cardiac event(e.g., a myocardial infarction).

In some embodiments, the ratio of two galectin-3 levels in a subject iscompared to a reference level that is a ratio of galectin-3 levelsmeasured in a subject (e.g., any of the control subjects describedherein or the same subject), for example, a reference level may be aratio of the levels of galectin-3 before and after onset of one or more(e.g., two, three, four, or five) disease (e.g., cardiac disease (e.g.,heart failure, coronary artery disease, cardiovascular disease, acutecoronary syndrome, and angina), renal insufficiency, stroke, orhypertension) symptoms; a ratio of the levels of galectin-3 before andafter diagnosis with disease (e.g., cardiac disease (e.g., heartfailure, coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension); aratio of the levels of galectin-3 before and after therapeutic treatmentfor a disease (e.g., cardiac disease (e.g., heart failure, coronaryartery disease, cardiovascular disease, acute coronary syndrome, andangina), renal insufficiency, stroke, or hypertension); a ratio of thegalectin-3 levels at two different time points during therapeutictreatment (e.g., inpatient or outpatient treatment) for a disease (e.g.,cardiac disease (e.g., heart failure, coronary artery disease,cardiovascular disease, acute coronary syndrome, and angina), renalinsufficiency, stroke, or hypertension); or a ratio of the galectin-3levels before and after a cardiac event (e.g., a myocardial infarction).

By the term “therapeutic treatment” or “treatment” is meant theadministration of one or more (e.g., two, three, or four) pharmaceuticalagents to a subject or the performance of a medical procedure on thebody of a subject (e.g., surgery, such as organ transplant or heartsurgery). Non-limiting examples of pharmaceutical agents that may beadministered to a subject include nitrates, calcium channel blockers,diuretics, thrombolytic agents, digitalis, renin-angiotensin-aldosteronesystem (RAAS) modulating agents (e.g., beta-adrenergic blocking agents,angiotensin-converting enzyme inhibitors, aldosterone antagonists, renininhibitors, and angiotensin II receptor blockers), andcholesterol-lowering agents (e.g., a statin). The term therapeutictreatment also include an adjustment (e.g., increase or decrease) in thedose or frequency of one or more (e.g., two, three, or four)pharmaceutical agents that a subject may be taking, the administrationof one or more (e.g., two, three, or four) new pharmaceutical agents tothe subject, or the removal of one or more (e.g., two, three, or four)pharmaceutical agents from the subject's treatment plan.

As used herein, a “subject” is a mammal, e.g., a human. In allembodiments, human nucleic acids, human polypeptides, and human subjectscan be used.

As used herein, a “biological sample” includes one or more of blood,serum, plasma, urine, and body tissue. In some embodiments, a sample isa serum or blood sample.

By the term “disease state” is meant the manifestation of one or more(e.g., at least two, three, four, or five) symptoms in a subject thatindicate either an abnormal decrease in the viability and/or biologicalactivity of one or more (e.g., at least two, three, four, or five)tissues in the body of the subject. Non-limiting examples of diseasestates in a subject include a cardiac disease (e.g., heart failure,heart attack, coronary artery disease, cardiovascular disease, acutecoronary syndrome, and angina), inflammation, stroke, renal failure,obesity, high cholesterol, and dyslipidemia.

By the phrase “physical symptoms associated with a disease state” ismeant the one or more (e.g., at least two, three, or four) symptoms thatare manifested by a subject having a particular disease state. Physicalsymptoms associated with several disease states are known in the art bymedical health professionals (e.g., physicians). Non-limiting examplesof physical symptoms associated with a cardiac disease (e.g., heartfailure, coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina) include shortness of breath, heart palpitations,increased heart rate, weakness, dizziness, nausea, sweating, chestdiscomfort or pressure, chest pain, arm pain, fullness, indigestion,sweating, wheezing, sleep apnea, or anxiety.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is two graphs depicting the data of a galectin-3 Kaplan-Meier(K-M) analysis for 1 year (FIG. 1A) or 4 years (FIG. 1B), which show thesurvival probability of subjects having low (below galectin-3 medianlevel, 0) or elevated levels of galectin-3 (greater than or equal togalectin-3 median level, 1).

FIGS. 2A and 2B, together, are two graphs depicting the data of ST2 K-Manalysis for 1 year (FIG. 2A) or 4 years (FIG. 2B), which show thesurvival probability of subjects having low (below soluble ST2 medianlevel, 0) or elevated levels of soluble ST2 (greater than or equal tosoluble ST2 median level, 1).

FIG. 2C is a graph depicting the data of a soluble ST2 K-M analysis,which shows the survival probability of subjects having low (below 35ng/mL, 0) or elevated levels of soluble ST2 (greater than or equal to 35ng/mL, 1).

FIG. 3 is a graph depicting the data from a soluble ST2 plus galectin-3K-M analysis, which shows the survival probability of subjects with bothsoluble ST2 and galectin-3 levels below median level (1), subjects witha soluble ST2 level below median level and a galectin-3 level greaterthan or equal to median level (2), subjects with a soluble ST2 levelgreater than or equal to soluble ST2 median level and a galectin-3 levelbelow galectin-3 median level (3), and subjects with a soluble ST2 levelgreater than or equal to median soluble ST2 levels and a galectin-3level greater than or equal to galectin-3 median level (4).

DETAILED DESCRIPTION

Provided are methods for evaluating the risk of an ACO in a subject(e.g., a human), deciding whether to initiate, terminate, or continuetreating a subject (e.g., treating on an inpatient basis), selecting asubject (e.g., a human) for participation in a clinical study, andselecting a subject (e.g., a human) for therapeutic treatment includingthe steps of determining (e.g., by measuring or assaying) a level of ST2in a biological sample from the subject and determining (e.g., bymeasuring or assaying) a level of galectin-3 in a biological sample fromthe subject. Kits for performing these methods are also provided.

Galectin-3

Galectin-3 is a member of the galectin family, which consists of animallectins that bind β-galactosides. Non-limiting examples of galectin-3protein include a proteins containing a sequence at least 90% identical(e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to thesequence of NCBI Accession Nos. NP_(—)001170859 or NP_(—)002297.Non-limiting examples of galectin-3 nucleic acids include nucleic acidscontaining a sequence at least 90% identical (e.g., at least 95%, 96%,97%, 98%, 99%, or 100% identical) to the sequence of NCBI Accession Nos.NM_(—)001177399.1 or NM_(—)002306.3.

Recently, a role for galectin-3 in the pathophysiology of heart failurehas been suggested (Sharma et al., Circulation 110:3121-3128 (2004)). Itwas observed that galectin-3 is specifically upregulated indecompensated heart failure compared with compensated heart failure inanimal models of heart failure. Galectin-3 has recently been proposed asa useful biomarker involved in the pathophysiology of heart failure (deBoer et al., Eur. J. Heart Failure 11:811-817 (2009)). Galectin-3 iswidely distributed throughout the body, including expression in heart,brain, and vessels (Yang et al., Expert Rev. Mol. Med. 13:e17-e39(2008)). Specifically, secretion of galectin-3 is associated withactivation of fibroblasts and fibrosis (Yang et al., supra).

Heart failure (HF) is a large medical and epidemiological problem, andrecent studies, both in acute and chronic HF, indicate that it isassociated with a high morbidity and mortality (Jessup et al.,Circulation 48:1217-1224 (2009)). Early identification of high-riskpatients may favorably affect outcome and biomarkers are increasinglybeing recognized to have important clinical value in this respect(Jessup et al., supra).

The first clinical study that evaluated the potential role of galectin-3as a plasma biomarker in acute heart failure was published by vanKimmenade et al. (J. Am. Coll. Cardiol. 48:1217-1224 (2006)). In thisstudy, 599 acutely dyspneic subjects were evaluated with the goal toestablish the usefulness of N-terminal prohormone brain natriureticpeptide (NT-proBNP), galectin-3, and apelin in diagnosing heart failureand predicting outcome. A blood sample was collected at baseline, andNT-proBNP, galectin-3, and apelin were measured in that sample. A totalof 209 patients in this cohort were diagnosed with heart failure. Inthis analysis, galectin-3 was not significant for diagnosis of heartfailure but was significant for prognosis in patients with heartfailure. For predicting short-term prognosis (60 days, primary end-pointall-cause mortality [n=17]), galectin-3 was the most powerful predictorwhen compared to NT-proBNP and apelin: an AUC for galectin-3 of 0.74(P=0.0001) and an AUC for NT-proBNP of 0.67 (P=0.009), with thedifference being borderline significant (P=0.05). In multivariateanalysis, galectin-3 was the strongest predictor for death within a 60day follow up period. Nevertheless, this study provides strong supportfor the exploration of galectin-3 as a biomarker that may predictprognosis, whereas its usefulness in detecting heart failure or addingincremental value (over currently used clinical correlates andNT-proBNP) in the diagnostic work-up of heart failure remains unclear.

A larger study in patients with chronic heart failure (n=232), showedthat galectin-3 predicts long-term outcome (mean follow-up, 3.4 y; HR,1.95; 95% CI, 1.24-3.09; P=0.004) (Lok et al., Clin. Res. Cardiol.99:323-328 (2010)). Because not many other biomarkers of heart failurewere measured, it is impossible to value the precise role of galectin-3in this cohort from this study.

Determining the level of galectin-3 in a subject typically includesobtaining a biological sample, e.g., plasma, serum, or blood, from thesubject. In some embodiments, levels of galectin-3 in the sample can bedetermined by measuring levels of polypeptide using methods known in theart and/or described herein, e.g., immunoassays, such as enzyme-linkedimmunosorbent assays (ELISA). One exemplary ELISA kit that is commercialavailable is the galectin-3 ELISA kit available from EMD Chemicals.Alternatively, levels of galectin-3 mRNA can be measured, again usingmethods known in the art and/or described herein, e.g., by quantitativePCR or Northern blotting analysis.

For example, a method as described herein, e.g., evaluating the risk ofan ACO in a subject, can include contacting a sample from a subject,e.g., a sample including blood, serum, or plasma, with a bindingcomposition (e.g., an antibody or oligonucleotide probe) thatspecifically binds to a polypeptide or nucleic acid of galectin-3. Themethods can also include contacting a sample from a control subject,normal subject, or normal tissue or fluid from the test subject, withthe binding composition, e.g., to provide a reference level ofgalectin-3.

An antibody that “binds specifically to” an antigen, bindspreferentially to the antigen in a sample containing other proteins. Theterm “antibody” as used herein refers to an immunoglobulin molecule orimmunologically active portion thereof, i.e., an antigen-bindingportion. Examples of immunologically active portions of immunoglobulinmolecules include F(ab) and F(ab′)2 fragments which can be generated bytreating the antibody with an enzyme such as pepsin. The antibody can bepolyclonal, monoclonal, recombinant, e.g., a chimeric or humanized,fully human, non-human, e.g., murine, monospecific, or single chainantibody. In some embodiments it has effector function and can fixcomplement. For the measurement of ST2, as further described below, anantibody produced from the hybridoma deposited at American Type CultureCollection and designated by Patent Deposit Designation PTA-10432 may beused.

An “oligonucleotide probe” (also referred to simply as a “probe”) is anucleic acid that is at least 10, and less than 200 (typically less thanabout 100 or 50) base pairs in length. A probe that “binds specificallyto” a target nucleic acid hybridizes to the target under high stringencyconditions. As used herein, the term “hybridizes under high stringencyconditions” describes conditions for hybridization and washing. As usedherein, high stringency conditions are 0.5 M sodium phosphate, 7% SDS at65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.Methods for performing nucleic acid hybridization assays are known tothose skilled in the art and can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

Detection can be facilitated by coupling (e.g., physically linking) theantibody or probe to a detectable substance (e.g., antibody labeling).Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride, quantum dots, or phycoerythrin; an example of a luminescentmaterial includes luminol; examples of bioluminescent materials includeluciferase, luciferin, and aequorin, and examples of suitableradioactive material include ¹²⁵I, ¹³¹I, ³⁵S, or ³H.

Diagnostic assays can be used with biological matrices such as livecells, cell extracts, cell lysates, fixed cells, cell cultures, bodilyfluids, or forensic samples. Conjugated antibodies useful for diagnosticor kit purposes, include antibodies coupled to dyes, isotopes, enzymes,and metals, see, e.g., Le Doussal et al., New Engl. J. Med. 146:169-175(1991); Gibellini et al., J. Immunol. 160:3891-3898 (1998); Hsing andBishop, New Engl. J. Med. 162:2804-2811 (1999); and Everts et al., NewEngl. J. Med. 168:883-889 (2002). Various assay formats exist, such asradioimmunoassays (RIA), enzyme-linked immunosorbent assay (ELISA), andlab on a chip (U.S. Pat. Nos. 6,176,962 and 6,517,234).

Known techniques in biochemistry and molecular biology can be used inthe methods described herein (see, e.g., Maniatis et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1982); Sambrook and Russell, Molecular Cloning, 3rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); Wu, Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif.(1993); and Ausbel et al., Current Protocols in Molecular Biology, Vols.1-4, John Wiley and Sons, Inc. New York, N.Y. (2001)).

Once a level of galectin-3 in a subject or sample has been determined,the level can be compared to a reference level. In some embodiments,e.g., where the level of galectin-3 is determined using an ELISA, thereference level will represent a threshold level, above which thesubject is identified as having an increased risk of an ACO, is selectedfor continued treatment on an inpatient basis, is selected forparticipation in a clinical study, or is selected for therapeutictreatment (as described herein). The reference level chosen may dependon the methodology (e.g., the particular antibody or ELISA kit) used tomeasure the levels of galectin-3.

Non-limiting threshold levels of galectin-3 may represent the medianlevel of galectin-3 in particular patient populations, e.g., subjectswith a BMI of less than 25, subjects with normal renal function,subjects without high risk cardiovascular disease, subjects with a BMIbetween 25 and 30, subjects with a BMI greater than 30, subjects with anelevated BMI, subjects with renal insufficiency, healthy men, healthywomen, and healthy children.

As noted above, a threshold level of galectin-3 may vary depending onthe methodology used to measure the levels of galectin-3. For example, athreshold level of galectin-3 measured using the ELISA kit from BenderMedsystems, Vienna, Austria may be between 1.0 to 3.0 ng/mL, 2.0 to 4.0ng/mL. 3.0 to 5.0 ng/mL, 4.0 to 6.0 ng/mL, 5.0 to 7.0 ng/mL, 6.0 to 8.0ng/mL, 7.0 to 9.0 ng/mL, 1.0 to 5.0 ng/mL, 5.0 to 9.0 ng/mL, 7.0 to 11.0ng/mL, or 9.0 to 13.0 ng/mL. Additional non-limiting examples ofgalectin-3 threshold levels include: 1.0 ng/mL, 1.1 ng/mL, 1.2 ng/mL,1.3 ng/mL, 1.4 ng/mL, 1.5 ng/mL, 1.6 ng/mL, 1.7 ng/mL, 1.8 ng/mL, 1.9ng/mL, 2.0 ng/mL, 2.1 ng/mL, 2.2 ng/mL, 2.3 ng/mL, 2.4 ng/mL, 2.5 ng/mL,2.6 ng/mL, 2.7 ng/nL, 2.8 ng/mL, 2.9 ng/mL, 3.0 ng/mL, 3.1 ng/mL, 3.2ng/mL, 3.3 ng/mL, 3.4 ng/mL, 3.5 ng/mL, 3.6 ng/mL, 3.7 ng/mL, 3.8 ng/mL,3.9 ng/mL, 4.0 ng/mL, 4.1 ng/mL, 4.2 ng/mL, 4.3 ng/mL, 4.4 ng/mL, 4.5ng/mL, 4.6 ng/mL, 4.7 ng/mL, 5.0 ng/mL, 5.2 ng/mL, 5.4 ng/mL. 5.6 ng/mL,5.8 ng/mL, 6.0 ng/mL, 6.2 ng/mL, 6.4 ng/mL, 6.6 ng/mL, 6.8 ng/mL, 7.0ng/mL, 7.2 ng/mL, 7.4 ng/mL, 7.6 ng/mL, 7.8 ng/mL, 8.0 ng/mL, 8.2 ng/mL,8.4 ng/mL, 8.6 ng/mL, 8.8 ng/mL, and 9.0 ng/mL.

A threshold level of galectin-3 measured using the ELISA kit from BGMedicine, Inc. include: greater than 25.9 ng/mL, 17.8 ng/mL to 25.9ng/mL, less than 17.8 ng/mL, 9 ng/mL, 10.0 ng/mL, 11.0 ng/mL, 12.0ng/mL, 13.0 ng/mL, 14.0 ng/mL, 15.0 ng/mL, 16.0 ng/mL, 17.0 ng/mL, 18.0ng/mL, 19.0 ng/mL, 20 ng/mL, 21 ng/mL, 22 ng/mL, 23 ng/mL, 24 ng/mL, 25ng/mL, and 26 ng/mL.

Additional threshold values are known (e.g., Sharma et al., Circulation110:3121-3128, (2004) and de Boer et al., Eur. J. Heart Failure11:811-817 (2009)) and can readily be determined by one skilled in theart. A threshold value of galectin-3 may reflect the level of galectin-3just below the level of galectin-3 observed in a subject presenting withone or more disease phenotypes (e.g., presenting with one or moresymptoms of a disease state, such as cardiac disease (e.g., heartfailure, coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, and hypertension).

Additional examples of a reference level of galectin-3 is a level ofgalectin-3 before or after onset of one or more (e.g., two, three, four,or five) disease (e.g., cardiac disease (e.g., heart failure, coronaryartery disease, cardiovascular disease, acute coronary syndrome, andangina), renal insufficiency, stroke, or hypertension) symptoms; a levelof galectin-3 before or after diagnosis with disease (e.g., cardiacdisease (e.g., heart failure, coronary artery disease, cardiovasculardisease, acute coronary syndrome, and angina), renal insufficiency,stroke, or hypertension); a level of galectin-3 before or aftertherapeutic treatment for a disease (e.g., cardiac disease (e.g., heartfailure, coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension); ora level of galectin-3 at a different time point during therapeutictreatment (e.g., inpatient or outpatient treatment) for a disease (e.g.,cardiac disease (e.g., heart failure, coronary artery disease,cardiovascular disease, acute coronary syndrome, and angina), renalinsufficiency, or hypertension); or before and after a cardiac event(e.g., a myocardial infarction).

In some embodiments, the level of galectin-3 is determined once, e.g.,at presentation. In some embodiments, the level of galectin-3 isdetermined at one or more of 2, 4, 6, 8, 12, 18, and/or 24 hours, and/or1-7 days after the onset of symptoms.

In some embodiments, the level of galectin-3 is determined more thanonce; in some embodiments, the higher measurement can be used. Inembodiments where the level of galectin-3 is determined more than once,the highest level can be used, or the change in levels (e.g., a ratio oftwo levels of galectin-3) can be determined and used.

In some embodiments, the ratio of two galectin-3 levels in a subject iscompared to a reference level that is a ratio of galectin-3 levelsmeasured in a subject (e.g., any of the control subjects describedherein or the same subject), for example, a reference level may be aratio of the levels of galectin-3 before and after onset of one or more(e.g., two, three, four, or five) disease (e.g., cardiac disease (e.g.,heart failure, coronary artery disease, cardiovascular disease, acutecoronary syndrome, and angina), renal insufficiency, stroke, orhypertension) symptoms; a ratio of the levels of galectin-3 before andafter diagnosis with disease (e.g., cardiac disease (e.g., heartfailure, coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension); aratio of the levels of galectin-3 before and after therapeutic treatmentfor a disease (e.g., cardiac disease (e.g., heart failure, coronaryartery disease, cardiovascular disease, acute coronary syndrome, andangina), renal insufficiency, stroke, or hypertension); a ratio of thegalectin-3 levels at two different time points during therapeutictreatment (e.g., inpatient or outpatient treatment) for a disease (e.g.,cardiac disease (e.g., heart failure, coronary artery disease,cardiovascular disease, acute coronary syndrome, and angina), renalinsufficiency, stroke, or hypertension); or a ratio of the galectin-3levels before and after a cardiac event (e.g., a myocardial infarction).

Levels of galectin-3 can also be determined multiple times to evaluate asubject's response to a treatment. For example, a level of galectin-3taken after administration of a treatment, e.g., one or more doses orrounds of a treatment, can be compared to levels of galectin-3 beforethe treatment was initiated, e.g., a baseline level, or at an early timepoint in ongoing treatment. The change in galectin-3 levels wouldindicate whether the treatment was effective; e.g., a reduction ingalectin-3 levels would indicate that the treatment was effective.

ST2

The ST2 gene is a member of the interleukin-1 receptor family, whoseprotein product exists both as a trans-membrane form, as well as asoluble receptor that is detectable in serum (Kieser et al., FEBS Lett.372(2-3):189-93 (1995); Kumar et al., J. Biol. Chem. 270(46):27905-13(1995); Yanagisawa et al., FEBS Lett. 302(1):51-3 (1992); Kuroiwa etal., Hybridoma 19(2):151-9 (2000)). ST2 was recently described to bemarkedly up-regulated in an experimental model of heart failure(Weinberg et al., Circulation 106(23):2961-6 (2002)), and preliminaryresults suggest that ST2 concentrations may be elevated in those withchronic severe HF (Weinberg et al., Circulation 107(5):721-6 (2003)) aswell as in those with acute myocardial infarction (MI) (Shimpo et al.,Circulation 109(18):2186-90 (2004)).

The transmembrane form of ST2 is thought to play a role in modulatingresponses of T helper type 2 cells (Lohning et al., Proc. Natl. Acad.Sci. U.S.A. 95(12):6930-6935 (1998); Schmitz et al., Immunity23(5):479-90 (2005)), and may play a role in development of tolerance instates of severe or chronic inflammation (Brint et al., Nat. Immunol.5(4):373-9 (2004)), while the soluble form of ST2 is up-regulated ingrowth stimulated fibroblasts (Yanagisawa et al., 1992, supra).Experimental data suggest that the ST2 gene is markedly up-regulated instates of myocyte stretch (Weinberg et al., 2002, supra) in a manneranalogous to the induction of the BNP gene (Bruneau et al., Cardiovasc.Res. 28(10):1519-25 (1994)).

Tominaga, FEBS Lett. 258:301-304 (1989), isolated murine genes that werespecifically expressed by growth stimulation in BALB/c-3T3 cells; theytermed one of these genes St2 (for Growth Stimulation-Expressed Gene 2).The St2 gene encodes two protein products: ST2 (IL1RL1), which is asoluble secreted form; and ST2L, a transmembrane receptor form that isvery similar to the interleukin-1 receptors. The HUGO NomenclatureCommittee designated the human homolog of ST2, the cloning of which wasdescribed in Tominaga et al., Biochim. Biophys. Acta. 1171:215-218(1992), as Interleukin 1 Receptor-Like 1 (IL1RL1). The two terms areused interchangeably herein.

The mRNA sequence of the shorter, soluble isoform of human ST2 can befound at GenBank Acc. No. NM_(—)003856.2, and the polypeptide sequenceis at GenBank Acc. No. NP_(—)003847.2; the mRNA sequence for the longerform of human ST2 is at GenBank Acc. No. NM_(—)016232.4; the polypeptidesequence is at GenBank Acc. No. NP_(—)057316.3. Additional informationis available in the public databases at GeneID: 9173, MIM ID #601203,and UniGene No. Hs.66. In general, in the methods described herein, thesoluble form of ST2 polypeptide is measured. Non-limiting examples ofsoluble ST2 protein include proteins containing a sequence at least 90%identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) tothe sequence of NCBI Accession Nos. NP_(—)003847.2. Non-limitingexamples of soluble ST2 nucleic acids include nucleic acids containing asequence at least 90% identical (e.g., at least 95%, 96%, 97%, 98%, 99%,or 100% identical) to the sequence of NCBI Accession Nos.NM_(—)003856.2.

Methods for detecting and measuring ST2 are known in the art, e.g., asdescribed in U.S. Pat. Pub. Nos. 2003/0124624, 2004/0048286, and2005/0130136, the entire contents of which are incorporated herein byreference. Kits for measuring ST2 polypeptide are also commerciallyavailable, e.g., the ST2 ELISA Kit manufactured by Medical & BiologicalLaboratories Co., Ltd. (MBL International Corp., Woburn, Mass.), no.7638. In addition, devices for measuring ST2 and other biomarkers aredescribed in U.S. Pat. Pub. No. 2005/0250156.

Levels of ST2 protein can also be measured using the antibodies producedfrom the hybridoma deposited at American Type Culture Collection anddesignated by Patent Deposit Designation PTA-10432, or any of theantibodies described in WO 2011/127412 and U.S. Patent ApplicationPublication No. 2011/0256635 (herein incorporated by reference).

Elevated concentrations of ST2 are markedly prognostic for death, with adramatic divergence in survival curves for those with elevated ST2 soonafter presentation, regardless of the underlying diagnosis. As oneexample, there is a dramatic relationship between elevations of ST2 andthe risk of mortality within four years following presentation withdyspnea. The relationship between ST2 and death in dyspneic patients wasindependent of diagnosis, and superseded all other biomarker predictorsof mortality in this setting, including other markers of inflammation,myonecrosis, renal dysfunction, and most notably NT-proBNP, a markerrecently described as having value for predicting death in thispopulation (Januzzi et al., Arch. Intern. Med. 166(3):315-320 (2006)).Indeed, most of the mortality in the study was concentrated amongsubjects with elevated ST2 levels at presentation; however, thecombination of elevated ST2 and NT-proBNP was associated with thehighest rates of death within one year.

In some embodiments, the level of ST2 is determined once, e.g., atpresentation. In some embodiments, the level of ST2 is determined at oneor more of 2, 4, 6, 8, 12, 18, and/or 24 hours, and/or 1-7 days afterthe onset of symptoms.

In some embodiments, the level of ST2 is determined more than once; insome embodiments, the higher measurement can be used. In embodimentswhere the level of ST2 is determined more than once, the highest levelcan be used, or the change in levels (e.g., a ratio of two levels ofST2) can be determined and used.

Levels of ST2 can also be determined multiple times to evaluate asubject's response to a treatment. For example, a level of ST2 takenafter administration of a treatment, e.g., one or more doses or roundsof a treatment, can be compared to levels of ST2 before the treatmentwas initiated, e.g., a baseline level, or at an early time point inongoing treatment. The change in ST2 levels would indicate whether thetreatment was effective; e.g., a reduction in ST2 levels would indicatethat the treatment was effective.

In some embodiments, the methods include determining the identity of thenucleotide sequence at RefSNP ID: rs1041973.

Once a level of ST2 has been determined in a subject, the level may becompared to a reference level. In some embodiments, e.g., where thelevel of ST2 is determined using an ELISA, the reference level willrepresent a threshold level, above which the subject is identified ashaving an increased risk of an ACO, selected for continued treatment onan inpatient basis, selected for participation in a clinical study, orselected for therapeutic treatment (as described herein). The referencelevel chosen may depend on the methodology (e.g., the particularantibody or ELISA kit) used to measure the levels of ST2. Referencelevels are known in the art and may readily be determined by one skilledin the art.

Non-limiting threshold levels of ST2 may represent the median level ofST2 in particular patient populations, e.g., subjects with a BMI of lessthan 25, subjects with normal renal function, subjects withoutcardiovascular disease, subjects with a BMI between 25 and 30, subjectswith a BMI greater than 30, subjects with an elevated BMI, subjects withrenal insufficiency, healthy men, healthy women, and healthy children.For example a threshold value for ST2 may fall within the range of about1.0 to 10 ng/mL, 5.0 ng/mL to 10 ng/mL, about 10.0 ng/mL to 20.0 ng/mL,about 10.0 ng/mL to 15.0 ng/mL, about 15.0 ng/mL to 20.0 ng/mL, about20.0 ng/ml to 40 ng/mL, about 20 ng/mL to 30 ng/mL, about 20 ng/mL to 25ng/mL, about 25 ng/mL to 30 ng/mL, about 30 ng/mL to about 40 ng/mL,about 30 ng/mL to 35 ng/mL, about 35 ng/mL to 40 ng/mL, about 40 ng/mLto about 60 ng/mL, about 40 ng/mL to about 50 ng/mL, and about 50 ng/mLto about 60 ng/mL.

In some embodiments, the threshold value for ST2 in men and women may beany value listed in the Table 1. For example, the threshold value of ST2in men may be 17.0 ng/mL, 18.0 ng/mL, 19.0 ng/mL, 20.0 ng/mL, 21.0ng/mL, 22.0 ng/mL, 23.0 ng/mL, 24.0 ng/mL, 25.0 ng/mL, 26.0 ng/mL, 27.0ng/mL, 28.0 ng/mL, 29.0 ng/mL, 30.0 ng/mL, 31.0 ng/mL, 32.0 ng/mL, 33.0ng/mL, 34.0 ng/mL, 35.0 ng/mL, 36.0 ng/mL, 37.0 ng/mL, 38.0 ng/mL, 39.0ng/mL, 40.0 ng/mL, 41.0 ng/mL, 42.0 ng/mL, 43.0 ng/mL, 44.0 ng/mL, 45.0ng/mL, 46.0 ng/mL, 47.0 ng/mL, 48.0 ng/mL, 49.0 ng/mL, and 50.0 ng/mL.Exemplary threshold values of ST2 in women may be 12.0 ng/mL, 13.0ng/mL, 14.0 ng/mL, 15.0 ng/mL, 16.0 ng/mL, 17.0 ng/mL, 18.0 ng/mL, 19.0ng/mL, 20.0 ng/mL, 21.0 ng/mL, 22.0 ng/mL, 23.0 ng/mL, 24.0 ng/mL, 25.0ng/mL, 26.0 ng/mL, 27.0 ng/mL, 28.0 ng/mL, 29.0 ng/mL, 30.0 ng/mL, 31.0ng/mL, 32.0 ng/mL, 33.0 ng/mL, 34.0 ng/mL, 35.0 ng/mL, 36.0 ng/mL, 37.0ng/mL, 38.0 ng/mL, 39.0 ng/mL, and 40.0 ng/mL.

TABLE 1 Serum ST2 Concentrations in Males and Females ST2 (ng/mL)Percentiles Combined Male Female 2.5 8.0 8.6 7.3 25 14.5 17.6 12.4 5018.8 23.6 16.2 75 25.3 30.6 19.9 90 34.3 37.2 23.7 95 37.9 45.4 29.097.5 45.6 48.5 33.1 99 50.2 52.7 39.9

As noted above, a threshold level of ST2 may vary depending on themethodology used to measure the levels of ST2. For example, if anantibody produced from the hybridoma deposited at American Type CultureCollection, designated with Patent Deposit Deposition PTA-10432 is usedto determine a ST2 level, non-limiting threshold values of ST2 mayinclude: below 20 ng/mL, 5 ng/mL to 15 ng/mL, 5.0 ng/mL to 10 ng/mL, 10ng/mL to 20 ng/mL, 10 ng/mL to 15 ng/mL, 14.5 ng/mL to 25.3 ng/mL, 15ng/mL to 25 ng/mL, 15 ng/mL to 20 ng/mL, 18.0 ng/mL to 20.0 ng/mL, 18.1ng/mL to 19.9 ng/mL, 20 ng/mL to 30 ng/mL, 20 ng/mL to 25 ng/mL, 25ng/mL to 35 ng/mL, 25 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 30 ng/mLto 35 ng/mL, 35 ng/mL to 45 ng/mL, 35 ng/mL to 40 ng/ml, and 40 ng/mL to45 ng/mL. Additional ST2 reference values that may be used when usingthe antibody produced from the hybridoma designated PTA-10432 is used todetermine a ST2 level include: for women, 12.4 ng/mL to 19.9 ng/mL, 12.0ng/mL to 20 ng/mL, 15.3 ng/mL to 17.4 ng/mL, 15.0 to 17.0 ng/mL, below20 ng/mL, and below 18 ng/mL; and for men, less than 31.0 ng/mL, lessthan 26.0 ng/mL, 17.6 ng/mL to 30.6 ng/mL, 17.0 ng/mL to 30.0 ng/mL,21.3 ng/mL to 25.1 ng/mL, and 21.0 ng/mL to 25.0 ng/mL. Additionalnon-limiting threshold values that may be used when a ST2 level ismeasured using the antibody produced from the hybridoma designatedPTA-10432 include: 10 ng/mL, 11 ng/mL, 12 ng/mL, 13 ng/mL, 14 ng/mL, 15ng/mL, 16 ng/mL, 17 ng/mL, 18 ng/mL, 19 ng/mL, 20 ng/mL, 21 ng/mL, 22ng/mL, 23 ng/mL, 24 ng/mL, 25 ng/mL, 26 ng/mL, 27 ng/mL, 28 ng/mL, 29ng/mL, 30 ng/mL, or 31 ng/mL.

In additional non-limiting examples, when a ST2 level is measured theST2 ELISA Kit (MBL International Corp., Woburn, Mass.), the thresholdlevels of ST2 include: 0.1 ng/mL to 0.6 ng/mL, 0.2 ng/mL to 0.6 ng/mL,0.2 ng/mL to 0.5 ng/mL, 0.3 ng/mL to 0.5 ng/mL, 0.2 ng/mL to 0.3 ng/mL,0.3 ng/mL to 0.4 ng/mL, and 0.4 ng/mL to 0.5 ng/mL. Additionalnon-limiting threshold values when using the ST2 ELISA Kit (MBLInternational Corp.) to measure a ST2 level include: 0.17 ng/mL, 0.18ng/mL, 0.19 ng/mL, 0.20 ng/mL, 0.21 ng/mL, 0.22 ng/mL, 0.23 ng/mL, 0.24ng/mL, 0.25 ng/mL, 0.26 ng/mL, 0.27 ng/mL, 0.28 ng/mL, or 0.29 ng/mL ofblood, serum, or plasma.

In some embodiments, the ratio of two ST2 levels in a subject iscompared to a reference level that is a ratio of ST2 levels measured ina subject (e.g., any of the control subjects described herein or thesame subject), for example, a reference level may be a ratio of thelevels of ST2 before and after onset of one or more (e.g., two, three,four, or five) disease (e.g., cardiac disease (e.g., heart failure,coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension)symptoms; a ratio of the levels of ST2 before and after diagnosis withdisease (e.g., cardiac disease (e.g., heart failure, coronary arterydisease, cardiovascular disease, acute coronary syndrome, and angina),renal insufficiency, stroke, or hypertension); a ratio of the levels ofST2 before and after therapeutic treatment for a disease (e.g., cardiacdisease (e.g., heart failure, coronary artery disease, cardiovasculardisease, acute coronary syndrome, and angina), renal insufficiency,stroke, or hypertension); a ratio of the ST2 levels at two differenttime points during therapeutic treatment (e.g., inpatient or outpatienttreatment) for a disease (e.g., cardiac disease (e.g., heart failure,coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension); ora ratio of the ST2 levels before and after a cardiac event (e.g., amyocardial infarction).

Methods of Evaluating the Risk of ACO

Methods of evaluating the risk of an ACO in a subject are provided. In aclinical setting, patients may present with a combination of ambiguoussymptoms, such that a physician or health care professional hasdifficulty in diagnosing the subject. In such situations, it isdifficult for the physician to determine whether the subject has anincreased risk of later experiencing an adverse clinical outcome.Provided are methods of evaluating the risk of an adverse clinicaloutcome in a subject requiring the steps of determining a level of ST2in a biological sample from the subject and determining a level ofgalectin-2 in a biological sample from the subject.

An adverse clinical outcome (ACO) may be an increase (e.g., by one, two,three, or four) in the number, severity, or frequency of one or more(e.g., at least two, three, or four) symptoms in a subject, death, ortreatment that is necessitated by the increase (e.g., by one, two,three, or four) in the number, severity, or frequency of one or more(e.g., at least two, three, or four) symptoms in a subject. An ACO maybe the rehospitalization, recurrence of one or more (e.g., at least two,three, or four) physical symptoms associated with a disease state, anincrease in the severity of one or more (e.g., at least two, three, orfour) physical symptoms associated with a disease state, an increase inthe frequency of one or more (e.g., at least two, three, or four)physical symptoms associated with a disease state, mortality, admissionto a health care facility, organ transplant (e.g., heart transplant), orsurgery (e.g., heart surgery). For example, the ACO may be an increasein the number, severity, duration, or frequency of one or more (e.g., atleast two, three, or four) symptoms associated with angina,cardiovascular disease, or heart failure. For patients presenting withcardiovascular disease, the ACO may be, for example, rehospitalizationor admission for cardiovascular disease or mortality.

The above methods may be performed on a subject presenting with one ormore (e.g., at least two, three, or four) symptoms in a health carefacility (e.g., hospital, such as in the emergency room). The method maybe performed by a physician, a laboratory technician, a physician'sassistant, or a nurse. A level of ST2 and galectin-3 may be measured ina biological sample from a patient within 14 days of the presentation ofthe patient to a health care facility (e.g., within 12 days, 10 days, 8days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 36 hours, 24hours, 20 hours, 16 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4hours, or 2 hours). The levels of ST2 and galectin-3 may also bemeasured in a biological sample from a subject that is alreadyhospitalized or under medical supervision (e.g., periodic check-ups orin an assisted care facility). The levels of ST2 and galectin-3 may alsobe measured in a biological sample that has been previously collectedfrom a subject.

Two different levels of ST2 and/or galectin-3 may be used to calculate aratio which may then be compared to a reference level (e.g., any of thereference ratios described above). For example, a ratio of the levels ofST2 and/or galectin-3 before and after onset of one or more (e.g., two,three, four, or five) disease (e.g., cardiac disease (e.g., heartfailure, coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension)symptoms; a ratio of the levels of ST2 and/or galectin-3 before andafter diagnosis with disease (e.g., cardiac disease (e.g., heartfailure, coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension); aratio of the levels of ST2 and/or galectin-3 before and aftertherapeutic treatment for a disease (e.g., cardiac disease (e.g., heartfailure, coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, or hypertension); a ratio ofthe levels of ST2 and/or galectin-3 at two different time points duringtherapeutic treatment (e.g., inpatient or outpatient treatment) for adisease (e.g., cardiac disease (e.g., heart failure, coronary arterydisease, cardiovascular disease, acute coronary syndrome, and angina),renal insufficiency, stroke, or hypertension); or a ratio of the ST2and/or galectin-3 levels before and after a cardiac event (e.g., amyocardial infarction) may be determined and compared to a referencevalue.

The subject may have been previously diagnosed with a cardiac disease(e.g., heart failure, heart attack, coronary artery disease,cardiovascular disease, acute coronary syndrome, and angina),inflammation, stroke, renal failure, obesity, high cholesterol, anddyslipidemia. The subject may also not have been previously diagnosed ashaving a disease (e.g., a cardiac disease (e.g., heart failure, heartattack, coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), inflammation, stroke, renal failure, obesity,high cholesterol, and dyslipidemia). In some embodiments of the methodsdescribed herein, the subject may present with one or more ambiguoussymptoms (e.g., shortness of breath, dizziness, discomfort, and nausea).The subject may have a BMI of between 25-29, a BMI of greater than orequal to 30, an elevated BMI, or have renal insufficiency.

The level of ST2 and galectin-3 may be measured in any biological sampleobtained from the subject. Non-limiting biological samples that may beused in the methods described herein include blood, serum, and plasma.The biological sample used to measure ST2 and the biological sample usedto measure galectin-3 may be gathered from the subject at the same time.The biological sample may be frozen or transported prior to determiningthe level of ST2 or galectin-3 present in the sample. Preferably, thebiological sample used to determine the level of ST2 or galectin-3 isserum.

Following the determination of a level of ST2 in a biological samplefrom the subject and the determination of a level of galectin-3 in abiological sample from the subject, the risk of adverse clinical outcomeis indicated by comparing the subject's levels of ST2 and galectin-3 toreference levels of ST2 and galectin-3. For example, the presence of anelevated level of ST2 (relative to a reference level of ST2) or thepresence of an elevated level of galectin-3 (relative to a referencelevel of galectin-3) indicates an increased risk (e.g., an increasedrisk of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%,160%, 170%, 180%, 190%, or 200%) of an ACO. In some embodiments, thepresence of both an elevated level of ST2 (relative to a reference levelof ST2) and an elevated level of galectin-3 (relative to a referencelevel of galectin-3) indicates a greatly increased risk (e.g., anincreased risk of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 140%, 160%, 180%, 200%, 220%,240%, 260%, 280%, or 300%) of an ACO. In some embodiments, anon-elevated level of ST2 and a non-elevated level of galectin-3indicates a decreased (e.g., a decreased risk of at least 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%) risk ofan ACO.

In additional examples, the presence of an elevated ratio of ST2 levels(relative to a reference ratio of ST2 levels) or the presence of anelevated ratio of galectin-3 levels (relative to a reference ratio ofgalectin-3 levels) indicates an increased risk (e.g., an increased riskof at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%,170%, 180%, 190%, or 200%) of an ACO. In some embodiments, the presenceof both an elevated ratio of ST2 levels (relative to a reference ratioof ST2 levels) and an elevated ratio of galectin-3 levels (relative to areference ratio of galectin-3 levels) indicates a greatly increased risk(e.g., an increased risk of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 140%, 160%, 180%,200%, 220%, 240%, 260%, 280%, or 300%) of an ACO. In some embodiments, anon-elevated ratio of ST2 levels (relative to a reference ratio of ST2levels) and a non-elevated ratio of galectin-3 levels (relative to areference ratio of galectin-3 levels) indicates a decreased risk (e.g.,an decreased risk of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, or 80%) of an ACO.

Any of the above described reference levels for ST2 or galectin-3 may beused in these methods. Any of the above described techniques fordetermining the level of ST2 or galectin-3 may be used in these methods.

The above methods may be used to determine the risk of an ACO within 1year and within 30 days of the time at which the biological sample wasobtained from the subject.

Methods for Deciding to Discharge or Continue Treatment on an InpatientBasis

Also provided are methods for deciding whether to discharge or initiate,terminate, or continue treating a subject on an inpatient basisincluding the steps of determining a level of ST2 in a biologicalsample, and determining a level of galectin-3 in a biological samplefrom the subject. This method may be performed by a health careprofessional (e.g., a physician, a physician's assistant, a nurse, or alaboratory technician).

Subjects often present to health care professionals with ambiguoussymptoms (e.g., shortness of breath, dizziness, nausea, or discomfort)that are difficult to diagnose. Often a health care profession has todecide whether to discharge the subject or whether to continue to treatthe subject on an inpatient basis (e.g., begin hospitalization, continuehospitalization, or admit to an assisted care facility). This method maybe performed when a subject presents himself or herself to a health careprofessional at a health care facility. This method may also beperformed on a subject who has already been admitted to a health carefacility (e.g., a hospital or an assisted care facility).

The method may be performed on subjects that have cardiac disease (e.g.,heart failure, coronary artery disease, cardiovascular disease, acutecoronary syndrome, and angina), renal insufficiency, stroke,inflammation, or hypertension, or on subjects that present with one ormore of the following symptoms: shortness of breath, heart palpitations,increased heart rate, weakness, dizziness, nausea, sweating, chestdiscomfort or pressure, chest pain, arm pain, fullness, indigestion,sweating, wheezing, sleep apnea, or anxiety. The method may also beperformed on patients with a BMI of 25-30, a BMI of greater than 30, oran elevated BMI.

The level of ST2 and the level of galectin-3 may be measured in any ofthe biological samples described above. The levels of ST2 and galectin-3may be measured using any of the methods described above and compared toany of the reference levels described above.

Following the determination of a level of ST2 in a biological samplefrom the subject and the determination of a level of galectin-3 in abiological sample from the subject, the subject's levels of ST2 andgalectin-3 relative to reference levels of ST2 and galectin-3 is used todetermine whether the subject should be discharged, should receivecontinued treatment (e.g., treatment on an inpatient basis) or whethertreatment should be initiated or terminated. For example, the presenceof an elevated level of ST2 (relative to a reference level of ST2) orthe presence of an elevated level of galectin-3 (relative to a referencelevel of galectin-3) indicates that the subject should receive continuedtreatment (e.g., treatment on an inpatient basis) or that treatmentshould be initiated. In some embodiments of the methods describedherein, the presence of both an elevated level of ST2 and an elevatedlevel of galectin-3 (relative to control levels) strongly indicates thatthe subject should receive continued treatment (e.g., treatment on aninpatient basis) or that treatment should be initiated. In additionalexamples, the presence of a non-elevated level of ST2 (relative to areference level of ST2) and the presence of a non-elevated level ofgalectin-3 (relative to a reference level of galectin-3) indicates thatthe subject should be discharged, receive treatment on an outpatientbasis, or that treatment should be terminated.

Two different levels of ST2 and/or galectin-3 may be used to calculate aratio which may then be compared to a reference level (e.g., a referenceratio as described above). For example, a ratio of the levels of ST2and/or galectin-3 before and after onset of one or more (e.g., two,three, four, or five) disease (e.g., cardiac disease (e.g., heartfailure, coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension)symptoms; a ratio of the levels of ST2 and/or galectin-3 before andafter diagnosis with disease (e.g., cardiac disease (e.g., heartfailure, coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension); aratio of the levels of ST2 and/or galectin-3 before and aftertherapeutic treatment for a disease (e.g., cardiac disease (e.g., heartfailure, coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension); aratio of the levels of ST2 and/or galectin-3 at two different timepoints during therapeutic treatment (e.g., inpatient or outpatienttreatment) for a disease (e.g., cardiac disease (e.g., heart failure,coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), renal insufficiency, stroke, or hypertension); ora ratio of the ST2 and/or galectin-3 levels before and after a cardiacevent (e.g., a myocardial infarction) may be determined and compared toa reference value.

In additional examples, the presence of an elevated ratio of ST2 levels(relative to a reference ratio of ST2 levels) or the presence of anelevated ratio of galectin-3 levels (relative to a reference ratio ofgalectin-3 levels) indicates that the subject should receive continuedtreatment (e.g., treatment on an inpatient basis) or that treatmentshould be initiated. In some embodiments of the methods describedherein, the presence of both an elevated ratio of ST2 levels (relativeto a reference ratio of ST2 levels) and an elevated ratio of galectin-3levels (relative to a reference ratio of galectin-3 levels) stronglyindicates that the subject should receive continued treatment (e.g.,treatment on an inpatient basis) or that treatment should be initiated.In some embodiments, a non-elevated ratio of ST2 levels (relative to areference ratio of ST2 levels) and a non-elevated ratio of galectin-3levels (relative to a reference ratio of galectin-3 levels) indicatesthat the subject should be discharged or that treatment should beterminated.

Continued treatment on an inpatient basis may mean new admission into ahealth care facility (e.g., a hospital or assisted care facility),continued admission in a health care facility (e.g., a hospital orassisted care facility), or frequent (e.g., daily, weekly, biweekly, ormonthly) consistent visits to a health care center (e.g., a clinic or ahospital). The subject may receive one or more (e.g., at least two,three, four, or five) pharmaceutical agents during the continuedtreatment or may be tested periodically for changes in the levels of ST2and/or galectin-3 in a biological sample from the subject. A decrease ina subject's ST2 and/or galectin-3 level relative to a reference sample(determined by subsequent testing) may later indicate that the subjectmay be discharged.

Methods for Selecting a Subject for Participation in a Clinical Study

Also provided are methods for selecting a subject for participation in aclinical study that include the steps of determining a level of ST2 in abiological sample in a subject and determining a level of galectin-3 ina biological sample in a subject.

Non-limiting examples of clinical studies include studies of cardiacdisease (e.g., heart failure, heart attack, coronary artery disease,cardiovascular disease, acute coronary syndrome, and angina),inflammation, stroke, renal failure, obesity, high cholesterol, anddyslipidemia. The clinical studies may also be used to study the effectof treatment of one or more (e.g., two, three, or four) pharmaceuticalagents (e.g., nitrates, calcium channel blockers, diuretics,thrombolytic agents, digitalis, renin-angiotensin-aldosterone system(RAAS) modulating agents (e.g., beta-adrenergic blocking agents,angiotensin-converting enzyme inhibitors, aldosterone antagonists, renininhibitors, and angiotensin II receptor blockers), andcholesterol-lowering agents (e.g., a statin)) on a subject.

The clinical studies may be performed by health care professionals(e.g., physicians, physician's assistants, nurses, phlebotomists, orlaboratory technicians) in a health care facility (e.g., a hospital, aclinic, or a research center). The biological samples may be obtainedfrom subjects present with one or more (e.g., at least two, three, four,or five) symptoms of a disease state (e.g., cardiovascular disease,angina, or heart failure), subjects that are admitted in a hospital, orsubjects who are asymptomatic.

The level of ST2 and the level of galectin-3 may be measured in any ofthe biological samples described above. The levels of ST2 and galectin-3may be measured using any of the methods described above and compared toany of the reference levels described above.

Following the determination of a level of ST2 in a biological samplefrom the subject and the determination of a level of galectin-3 in abiological sample from the subject, a subject is selected forparticipation in a clinical study based on the comparison of subject'slevels of ST2 and galectin-3 to reference levels of ST2 and galectin-3.For example, the subject is selected for participation in a clinicalstudy if the subject has an elevated level of ST2 (relative to areference level of ST2) or an elevated level of galectin-3 (relative toa reference level of galectin-3). In some embodiments of the methodsdescribed herein, the subject is strongly selected for participation ina clinical study if the subject has both an elevated level of ST2(relative to a reference level of ST2) and an elevated level ofgalectin-3 (relative to a reference level of galectin-3). In someembodiments, the subject is excluded from participation in a clinicalstudy if the subject has both a non-elevated level of ST2 (relative to areference level of ST2) and a non-elevated level of galectin-3 (relativeto a reference level of galectin-3).

In another embodiment, a subject with a level of galectin-3 that islower than a reference level of galectin-3 (e.g., a subject having anelevated level of ST2 compared to a reference level and a decreasedlevel of galectin-3 relative to a reference level) may be selected forparticipation in a clinical study (e.g., a study of the effect of one ormore (e.g., at least two, three, or four) statins in a subject (e.g., asubject with a cardiac disease (e.g., heart failure, heart attack,coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), inflammation, stroke, renal failure, obesity,high cholesterol, and/or dyslipidemia).

As described above, two different levels of ST2 and/or galectin-3 may beused to calculate a ratio which may then be compared to a referencelevel (e.g., a reference ratio as described above). For example, a ratioof the levels of ST2 and/or galectin-3 before and after onset of one ormore (e.g., two, three, four, or five) disease (e.g., a cardiac disease(e.g., heart failure, heart attack, coronary artery disease,cardiovascular disease, acute coronary syndrome, and angina),inflammation, stroke, renal failure, obesity, high cholesterol, anddyslipidemia), renal insufficiency, and hypertension) symptoms; a ratioof the levels of ST2 and/or galectin-3 before and after diagnosis withdisease (e.g., a cardiac disease (e.g., heart failure, heart attack,coronary artery disease, cardiovascular disease, acute coronarysyndrome, and angina), inflammation, stroke, renal failure, obesity,high cholesterol, and dyslipidemia); a ratio of the levels of ST2 and/orgalectin-3 before and after therapeutic treatment for a disease (e.g., acardiac disease (e.g., heart failure, heart attack, coronary arterydisease, cardiovascular disease, acute coronary syndrome, and angina),inflammation, stroke, renal failure, obesity, high cholesterol, anddyslipidemia); a ratio of the levels of ST2 and/or galectin-3 at twodifferent time points during therapeutic treatment (e.g., inpatient oroutpatient treatment) for a disease (e.g., a cardiac disease (e.g.,heart failure, heart attack, coronary artery disease, cardiovasculardisease, acute coronary syndrome, and angina), inflammation, stroke,renal failure, obesity, high cholesterol, and dyslipidemia); or a ratioof the ST2 and/or galectin-3 levels before and after a cardiac event(e.g., a myocardial infarction) may be determined and compared to areference value.

In some examples of the methods described herein, a subject is selectedfor participation in a clinical study if the subject has an elevatedratio of ST2 levels (relative to a reference ratio of ST2 levels) or anelevated ratio of galectin-3 levels (relative to a reference ratio ofgalectin-3 levels). In some examples of the methods described herein, asubject is strongly selected for participation in a clinical study ifthe subject has an elevated ratio of ST2 levels (relative to a referenceratio of ST2 levels) and an elevated ratio of galectin-3 levels(relative to a reference ratio of galectin-3 levels). In someembodiments, a subject is excluded from participation in a clinicalstudy if the subject has a non-elevated ratio of ST2 levels (relative toa reference ratio of ST2 levels) and a non-elevated ratio of galectin-3levels (relative to a reference level of galectin-3 levels).

Additional factors may further indicate that the subject should beincluded in a clinical study. These additional factors include priordiagnosis with cardiovascular disease, angina, heart attack, heartfailure, renal failure, inflammation, or stroke, or presentation of oneor more (e.g., two, three, or four) of the following symptoms: shortnessof breath, heart palpitations, increased heart rate, weakness,dizziness, nausea, sweating, chest discomfort or pressure, chest pain,arm pain, fullness, indigestion, sweating, wheezing, sleep apnea, andanxiety. Additional factors include a BMI of 25-30, a BMI of greaterthan 30, an elevated BMI, or continued therapy on one or more (e.g., atleast two, three, four, or five) pharmaceutical agents (e.g., nitrates,calcium channel blockers, diuretics, thrombolytic agents, digitalis,renin-angiotensin-aldosterone system (RAAS) modulating agents (e.g.,beta-adrenergic blocking agents, angiotensin-converting enzymeinhibitors, aldosterone antagonists, renin inhibitors, and angiotensinII receptor blockers), and cholesterol-lowering agents (e.g., astatin)).

Methods for Selecting a Therapeutic Treatment for a Subject

Also provided are methods for selecting a treatment for a subjectrequiring the steps of determining a level of ST2 in a biological samplefrom the subject and determining a level of galectin-3 in a biologicalsample from the subject.

This method may be performed on subjects that present clinically (e.g.,diagnosed) with one or more (e.g., at least two, three, four, or five)symptoms of a disease (e.g., a cardiac disease (e.g., heart failure,heart attack, coronary artery disease, cardiovascular disease, acutecoronary syndrome, and angina), inflammation, stroke, renal failure,obesity, high cholesterol, and dyslipidemia). The method may also beperformed on subjects that present with one or more (e.g., two, three,or four) of the following symptoms: shortness of breath, heartpalpitations, increased heart rate, weakness, dizziness, nausea,sweating, chest discomfort or pressure, chest pain, arm pain, fullness,indigestion, sweating, wheezing, sleep apnea, and anxiety. The subjectmay have been previously diagnosed one or more (e.g., two, three, four,or five) of the following conditions: a cardiac disease (e.g., heartfailure, heart attack, coronary artery disease, cardiovascular disease,acute coronary syndrome, and angina), inflammation, stroke, renalfailure, obesity, high cholesterol, and dyslipidemia, or the subject maynot have been previously diagnosed with a disease. The subject may havebeen previously admitted to a health care facility and previouslydischarged, or may be a patient admitted in a health care facility(e.g., hospital or assisted care facility). The methods may be performedby a health care professional (e.g., a physician, a physician'sassistant, a nurse, or a laboratory technician) in a health carefacility (e.g., a hospital, clinic, or assisted care facility).

The level of ST2 and the level of galectin-3 may be measured in any ofthe biological samples described above. The levels of ST2 and galectin-3may be measured using any of the methods described above and compared toany of the reference levels described above.

Following the determination of a level of ST2 in a biological samplefrom the subject and the determination of a level of galectin-3 in abiological sample from the subject, the subject's levels of ST2 andgalectin-3 relative to reference levels of ST2 and galectin-3 is used toselect a treatment for the subject. For example, the presence of anelevated level of ST2 (relative to a reference level of ST2) or thepresence of an elevated level of galectin-3 (relative to a referencelevel of galectin-3) is used to select a treatment for the subject. Insome embodiments of the methods described herein, the presence of bothan elevated level of ST2 and an elevated level of galectin-3 (relativeto control levels) is predominantly (strongly) used to select thetherapeutic treatment for the subject. In some embodiments of themethods described herein, the presence of both a non-elevated level ofST2 and a non-elevated level of galectin-3 (relative to control levels)is used to select a therapeutic treatment for the subject.

As described above, two different levels of ST2 and/or galectin-3 may beused to calculate a ratio which may then be compared to a referencelevel (e.g., a reference ratio as described above). For example, a ratioof the levels of ST2 and/or galectin-3 before and after onset of one ormore (e.g., two, three, four, or five) disease (e.g., a cardiac disease(e.g., heart failure, heart attack, coronary artery disease,cardiovascular disease, acute coronary syndrome, and angina),inflammation, stroke, renal failure, obesity, high cholesterol, anddyslipidemia) symptoms; a ratio of the levels of ST2 and/or galectin-3before and after diagnosis with disease (e.g., a cardiac disease (e.g.,heart failure, heart attack, coronary artery disease, cardiovasculardisease, acute coronary syndrome, and angina), inflammation, stroke,renal failure, obesity, high cholesterol, and dyslipidemia); a ratio ofthe levels of ST2 and/or galectin-3 before and after therapeutictreatment for a disease (e.g., a cardiac disease (e.g., heart failure,heart attack, coronary artery disease, cardiovascular disease, acutecoronary syndrome, and angina), inflammation, stroke, renal failure,obesity, high cholesterol, and dyslipidemia); a ratio of the levels ofST2 and/or galectin-3 at two different time points during therapeutictreatment (e.g., inpatient or outpatient treatment) for a disease (e.g.,a cardiac disease (e.g., heart failure, heart attack, coronary arterydisease, cardiovascular disease, acute coronary syndrome, and angina),inflammation, stroke, renal failure, obesity, high cholesterol, anddyslipidemia); or a ratio of the ST2 and/or galectin-3 levels before andafter a cardiac event (e.g., a myocardial infarction) may be determinedand compared to a reference value.

In some examples of the methods described herein, the presence of anelevated ratio of ST2 levels (relative to a reference ratio of ST2levels) or the presence of an elevated ratio of galectin-3 levels(relative to a reference ratio of galectin-3 levels) is used to select atreatment for a subject. In some examples of the methods describedherein, the presence of both an elevated ratio of ST2 levels (relativeto a reference ratio of ST2 levels) and an elevated ratio of galectin-3levels (relative to a reference ratio of galectin-3 levels) is stronglyused to select a treatment for a subject. In some embodiments, thepresence of a non-elevated ratio of ST2 levels (relative to a referenceratio of ST2 levels) and the presence of a non-elevated ratio ofgalectin-3 levels (relative to a reference level of galectin-3 levels)is used to select a treatment for a subject.

Additional factors may further be used to select a therapeutic treatmentfor the subject. These additional factors include prior diagnosis withone or more (e.g., two, three, four, or five) of the followingconditions: cardiovascular disease, angina, heart attack, heart failure,renal failure, inflammation, and stroke, and/or presentation of one ormore (e.g., two, three, or four) of the following symptoms: shortness ofbreath, heart palpitations, increased heart rate, weakness, dizziness,nausea, sweating, chest discomfort or pressure, chest pain, arm pain,fullness, indigestion, sweating, wheezing, sleep apnea, or anxiety.Additional factors include a BMI of 25-30, a BMI of greater than 30, anelevated BMI, or continued therapy on one or more (e.g., at least two,three, four, or five) pharmaceutical agents (e.g., nitrates, calciumchannel blockers, diuretics, thrombolytic agents, digitalis,renin-angiotensin-aldosterone system (RAAS) modulating agents (e.g.,beta-adrenergic blocking agents, angiotensin-converting enzymeinhibitors, aldosterone antagonists, renin inhibitors, and angiotensinII receptor blockers), and cholesterol-lowering agents (e.g., astatin)). Examples of these pharmaceutical agents are well known in theart.

The therapeutic treatment may be the administration of one or more(e.g., two, three, or four) pharmaceutical agents to the subject and/orthe performance of a medical procedure on the body of the subject (e.g.,surgery, such as organ transplant or heart surgery). Non-limitingexamples of pharmaceutical agents that may be administered to a subjectinclude nitrates, calcium channel blockers, diuretics, thrombolyticagents, digitalis, renin-angiotensin-aldosterone system (RAAS)modulating agents (e.g., beta-adrenergic blocking agents,angiotensin-converting enzyme inhibitors, aldosterone antagonists, renininhibitors, and angiotensin II receptor blockers), andcholesterol-lowering agents (e.g., a statin). In another example, thetherapeutic treatment may be an adjustment (e.g., an increase ordecrease) in the dose, duration, or frequency of one or more (e.g., atleast two, three, or four) pharmaceutical agents that a subject may betaking, the administration of one or more (e.g., at least two, three, orfour) new pharmaceutical agents to the subject, or the removal of one ormore (e.g., at least two, three, or four) pharmaceutical agents from thesubject's treatment plan.

These methods may be repeated over time (e.g., weekly, biweekly,monthly, once every two months, once every six months, once a year) toselect a different therapeutic treatment for the subject.

Additional Markers

Some embodiments of all of the above methods, may further includedetermining the level of one or more (e.g., at least two, three, four,or four) additional markers in a biological sample from the subject. Theadditional markers may be selected from the group of: proANP, NT-proANP,ANP, proBNP, NT-proBNP, BNP, troponin, CRP, creatinine, Blood UreaNitrogen (BUN), liver function enzymes, albumin, and bacterialendotoxin. The one or more additional markers can be measured in any ofthe biological samples described above. The presence of an increasedlevel (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%,150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%,270%, 280%, 290%, or 300%) of one or more (e.g., at least two, three, orfour) of proANP, NT-proANP, ANP, proBNP, NT-proBNP, BNP, troponin, CRP,creatinine, Blood Urea Nitrogen (BUN), liver function enzymes, albumin,and bacterial endotoxin in a subject compared to a reference level foreach of these markers may further indicate that the subject has anincreased risk of an ACO, the subject should receive continued treatment(e.g., treatment on an inpatient basis) or that treatment should beinitiated or terminated, the subject should be selected forparticipation in a clinical study, or the subject should be selected fortreatment. The reference levels of these additional markers may be athreshold value or may be the level of these markers in a patientpopulation, e.g., subjects with a BMI of less than 25, subjects withnormal renal function, subjects without cardiovascular disease, subjectswith a BMI between 25 and 30, subjects with a BMI greater than 30,subject with an elevated BMI, subjects with renal insufficiency, healthymen, healthy women, and healthy children. Preferably, the above methodsfurther include determining the level of BNP.

Methods for determining the levels of these additional markers are knownin the art. Commercial kits for determining these additional markers areavailable.

Kits

Also provided are kits containing an antibody that specifically binds toST2, an antibody that specifically binds to galectin-3, and instructionsfor using the kit (e.g., the antibodies in the kit) to perform any ofthe methods described herein. The antibody that specifically binds ST2and the antibody that specifically binds to galectin-3 may bepolyclonal, monoclonal, recombinant, e.g., a chimeric or humanized,fully human, non-human, e.g., murine, monospecific, or single chainantibody. Any of the kits described herein may also be provided as anELISA assay (e.g., may further include one or more secondary antibodiesand/or a substrate for detection). For example, any of the kitsdescribed herein may include an antibody produced from the hybridomadeposited at American Type Culture Collection and designated by PatentDeposit Designation PTA-10432. Additional examples of antibodies thatspecifically bind to ST2 are described in WO 2011/127412 and U.S. PatentApplication Publication No. 2011/0256635 (herein incorporated byreference).

Any of the kits described herein may also include one or more (e.g.,two, three, four, or five) additional antibodies for one or more (e.g.,two, three, four, or five) additional markers selected from the groupof: proANP, NT-proANP, ANP, proBNP, NT-proBNP, BNP, troponin, CRP,creatinine, liver function enzymes, albumin, and bacterial endotoxin.Antibodies for ST2, galectin-3, proANP, NT-proANP, ANP, proBNP,NT-proBNP, BNP, troponin, CRP, creatinine, liver function enzymes,albumin, and bacterial endotoxin are commercially available.

The invention is further described in the following example, which doesnot limit the scope of the invention described in the claims.

EXAMPLE

It has been reported that circulating concentrations of both theinterleukin-1 receptor like 1 family member ST2 and galectin-3 areelevated in patients with heart failure (HF) and are independentlyprognostic. Experiments were performed to investigate the utility of ST2measurement in patients with elevated concentrations of galectin-3.

The subjects used in this Example took part in the ProBNP Investigationof Dyspnea in the Emergency Department (PRIDE) Study, a prospective,blinded study of 599 dyspneic subjects presenting to the ED of theMassachusetts General Hospital, which was performed for the purpose ofvalidating the diagnostic and prognostic use of NT-proBNP testing. Theresults of the PRIDE study were recently reported (Januzzi et al., Am.J. Cardiol. 95(8):948-954 (2005)).

The gold standard for the diagnosis of acute HF was based on theimpression of reviewing physicians, blinded to NT-proBNP values, who hadall available information from presentation through a 60-day follow-upperiod; for the few patients in whom a diagnosis was uncertain, thereviewers were instructed to utilize the guidelines as reported by theFramingham Heart Study (McKee et al., N. Engl. J. Med. 285(26):1441-1446(1971)).

As reported, 209 subjects (35%) in the PRIDE study were adjudicated tohave dyspnea due to acute destabilized HF, of whom 17 had mild (ClassII) symptoms by the New York Heart Association (NYHA) classification, 80had moderate (Class III) symptoms, and 112 had severe (Class IVsymptoms).

At the end of one year, the managing physician for each patient wascontacted for the purposes of ascertainment of vital status. Asreported, follow-up at one year was complete in 597 subjects overall.

Blood collected at the time of presentation was later analyzed forconcentrations of ST2, using an enzyme-linked immunosorbent assay(Critical Diagnostics, San Diego), as described herein. This assayutilizes monoclonal antibodies to human ST2 for both capture anddetection, and had an inter-assay coefficient of variation of <10% inthe present analysis. Galectin-3 was analyzed using a commerciallyavailable enzyme-linked immunosorbent assay kit (Bender Medsystems,Vienna, Austria) and was measured on a Victor 2 plate reader (PerkinElmer, Turku, Finland). Calibration of the assay was performed accordingto the manufacturer's protocol. Values were normalized to a standardcurve. The intra-assay and inter-assay variances for galectin-3 were5.6% and 8.6%, respectively. The blood used for the present study hadbeen previously subjected to a single freeze-thaw cycle.

Distribution of Analyte Values

In this cohort of 209 patients with heart failure galectin-3 values wereproduced from 200 patients and soluble ST2 values were produced from 204patients. Both analytes showed a non-normal distribution so subsequentrisk analysis models will be based on either median values, a valuecorresponding to the upper percentile of normal or as log (natural log)transformed, continuous variables (Table 2). As shown in Table 1, aconcentration for soluble ST2 of 35 ng/mL is above the 90th percentileof normal.

TABLE 2 Analyte Median and IQR values N Median (ng/mL) 25-75 P NormalDistr. Galectin-3 200 9.2  7.4-12.0 <0.0001 ST2 204 42.7 26.9-78.7<0.0001

Risk Analysis

In this study cohort both analytes, soluble ST2 and galectin-3, weresignificant predictors of risk of mortality within 1 year as evaluatedin a Cox proportional hazards regression model with each analyte used asa log transformed, continuous variable (Tables 3A and 3B). In thisanalysis, soluble ST2 has a higher (stronger) hazard ratio (HR), butboth are statistically significant.

TABLE 3A Univariate Cox HR Model; Mortality Within 1 Year Covariate P HR95% CI of HR Ln ST2 <0.0001 2.12 1.58 to 2.85 Ln Galectin-3 0.0249 1.781.08 to 2.94

TABLE 3B Univariate Cox HR Model; Mortality Within 4 Years Covariate PHR 95% CI of HR Ln ST2 0.0001 1.53 1.23 to 1.91 Ln Galectin-3 0.00891.63 1.13 to 2.36

In a multivariate Cox proportional hazards regression model with eachanalyte used as a log transformed, continuous variable both analytes aresignificant for risk of mortality within 4 years, however galectin-3 isno longer statistically significant for risk of mortality within 1 year,while soluble ST2 remains strongly predictive (Tables 4A and 4B).

TABLE 4A Multivariate Cox HR Model; Mortality Within 1 Year Covariate PHR 95% CI of HR Ln ST2 <0.0001 2.07 1.54 to 2.79 Ln Galectin-3 0.08181.63 0.94 to 2.81

TABLE 4B Multivariate Cox HR Model; Mortality Within 4 Years Covariate PHR 95% CI of HR Ln ST2 0.0006 1.49 1.19 to 1.86 Ln Galectin-3 0.03211.52 1.04 to 2.23

Both analytes were also tested for significance to predict risk in a 30day follow up period. As was observed for the longer 1-year follow upperiod, both analytes are significant predictors of all-cause mortalityin a univariate Cox proportional hazards regression model with eachanalyte used as a log transformed, continuous variable (Table 5).

TABLE 5 Univariate Cox HR Model; Mortality Within 30 Days Covariate P HR95% CI of HR Ln ST2 0.0001 3.50 1.84 to 6.65 Ln Galectin-3 0.0030 3.411.52 to 7.61

In the shorter follow up model, galectin-3 retains significance whensoluble ST2 is also included in the model (Table 6).

TABLE 6 Multivariate Cox HR Model; Mortality Within 30 Days Covariate PHR 95% CI of HR Ln ST2 0.0003 3.27 1.73 to 6.20 Ln Galectin-3 0.01483.34 1.27 to 8.77

Each analyte was also evaluated for risk of all-cause mortality within 1year and 4 years by Kaplan-Meier (K-M) analysis using the cohort medianvalue for each. As shown in FIGS. 1A and 1B, when analyzed by the medianconcentration value galectin-3 is not a significant predictor ofall-cause mortality risk within 1 year but does reach significance over4 years. However, as shown in FIGS. 2A and 2B, soluble ST2 is a stronglysignificant predictor of all-cause mortality risk within both 1 year and4 years.

ST2 was also evaluated by K-M analysis using the lower concentration of35 ng/mL (FIG. 2C). As shown in FIG. 2C, soluble ST2 is a stronglysignificant predictor of mortality within 4 years.

To determine whether soluble ST2 adds predictive value in galectin-3heart failure patients a subset of patients with galectin-3concentrations greater than or equal to the cohort median concentrationof 9.2 ng/mL were tested with soluble ST2. Table 7 shows the resultsfrom univariate Cox proportional hazards regression models with solubleST2 used as a log transformed, continuous variable for assessment ofall-cause mortality risk within 1 year and within 30 days. Soluble ST2is strongly predictive in both time frames in galectin-3 heart failurepatients.

TABLE 7 ST2 additive in galectin-3 HF patients (N = 98 patients)Covariate P HR 95% CI of HR Ln ST2 (1 year) 0.0010 1.96 1.32 to 2.93 LnST2 (30 days) 0.0029 3.25 1.50 to 7.04

The opposite is not completely true however (see Tables 8A and 8B). Inthe subset of soluble ST2 heart failure patients, patients with solubleST2 concentrations greater than or equal to the cohort medianconcentration of 42.7 ng/mL, or at the lower soluble ST2 concentrationof 35 ng/mL, galectin-3 is not a significant predictor of all-causemortality within 1 year, but does reach statistical significance forrisk within 30 days as evaluated by Cox proportional hazards regressionmodels (Tables 8A and 8B).

TABLE 8A Galectin-3 additive to ST2 HF patients (ST2 ≧ median), N = 98Covariate P HR 95% CI of HR Ln Galectin-3 (1 year) 0.2251 1.59 0.75 to3.36  Ln Galectin-3 (30 days) 0.0214 4.10 1.24 to 13.53

TABLE 8B Galectin-3 additive to ST2 HF patients (ST2 ≧ 35 ng/mL), N =122 Covariate P HR 95% CI of HR Ln Galectin-3 (1 year) 0.4332 1.26 0.71to 2.26 Ln Galectin-3 (30 days) 0.0467 2.44 1.02 to 5.86

These results are also illustrated in FIG. 3, and summarized in Table 9,where a K-M curve analysis of this cohort when the two analytes arecombined (log rank p=0.0011). In this analysis in patients withconcentrations below the median for both analytes the lowest riskprofile is observed (line 1). When soluble ST2 is below median andgalectin-3 is ≧median risk increases modestly (line 2). When galectin-3is below median and soluble ST2 is ≧median risk increases significantly(line 3). And in patients with concentrations≧median for both analytesrisk is the greatest.

TABLE 9 Summary of K-M Analysis Shown in FIG. 3 Factor/Bin 1 2 3 4 N 6243 45 57 N decedant  8 11 17 24 % decedant 12.9% 25.6% 37.8% 42.1%

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method for evaluating the risk of an adverseclinical outcome (ACO) in a subject, the method comprising: a)determining a level of soluble ST2 in a biological sample from thesubject; and b) determining a level of galectin-3 in a biological samplefrom the subject, wherein the subject's levels of soluble ST2 andgalectin-3 relative to reference levels of soluble ST2 and galectin-3indicate the subjects's risk of an ACO.
 2. A kit comprising: i) anantibody that specifically binds to soluble ST2; ii) an antibody thatspecifically binds to galectin-3; and iii) instructions for using thekit in the method of claim 1.